The present invention is directed to the use of recombinant DNA techniques to confer upon microorganism host cells the capacity for selected bioconversions. More specifically, the invention is directed to the cloning of toluene monooxygenase genes from a newly isolated and characterized Pseudomonas strain, Pseudomonas mendocina KR-1. The present invention thus provides genetically engineered microorganisms that over produce toluene monooxygenase enzymes and proteins, and therefore provides more efficient means of conducting bioconversions dependent on this enzyme system.
Recently, a bacterium identified as Pseudomonas mendocina KR-1 (PmKR1) was isolated by Richardson and Gibson from an algal-mat taken from a fresh water lake. Whited, Ph. D. Dissertation, The University of Texas at Austin, Library Reference No. W586 (1986). PmKR1 utilizes toluene as a sole carbon and energy source. Other strains of Pseudomonas have been previously isolated and described which metabolize or degrade toluene, including Pseudomonas putida mt-2 (Pp mt-2). Williams and Murry, J. Bacteriol. 120: 416-423 (1974) and Pseudomonas putida PpF1 (PpF1) (Gibson, et al. Biochemistry 9:1626-1630 (1970). However, the genes, the enzymes and the pathways for toluene metabolism in these various Pseudomonas strains are distinct and non-overlapping.
The catabolic pathway for the degradation of toluene by Pp mt-2 has been designated TOL. The genes for the TOL pathway are encoded on isofunctional catabolic plasmids found in certain strains of Pseudomonas. The reference plasmid for the TOL degradative pathway is pWWO originally isolated from Pp mt-2. The genetics and biochemistry of the TOL pathway are well described. Kunz and Chapman, J. Bacteriol. 146:179-191 (1981); Williams and Murry, J. Bacteriol. 120:416-423 (1974); Williams and Worsey, J. Bacteriol. 125:818-828 (1976); Worsey and Williams, J. Bacteriol. 124:7-13 (1975); Murry, et al., Eur. J. Biochem. 28:301-310 (1972). A brief summary of the TOL pathway is as follows: initial attack of toluene is at the methyl group which undergoes successive oxidations to form benzoic acid, which is further oxidized by formation of a cis-carboxylic acid diol, which is oxidized to form catechol, which is then degraded by enzymes of a meta cleavage pathway to acetaldehyde and pyruvate.
A second catabolic pathway for the degradation of toluene by PpF1 has been established and designated TOD. In contrast to the TOL pathway, the genes for the TOD pathway are located on the bacterial chromosome and are not plasmid-encoded. Finette, et al., J. Bacteriol. 160:1003-1009 (1984); Finette, Ph. D. Dissertation, The University of Texas at Austin, Library Reference No. F494 (1984). The genetics and biochemistry of the TOD pathway has been studied by Finette, et al. (supra); Finette (supra); Gibson, et al. Biochemistry 9:1626-1630 (1970); Kobal, et al., J. Am. Chem. Soc. 95:4420-4421 (1973); Ziffer, et al., J. Am. Chem. Soc. 95:4048-4049 (1973); Dagley, et al., Nature 202:775-778 (1964); Gibson, et al., Biochemistry 7:2653-2662 (1968). A brief summary of the TOD pathway is as follows: the initial attack of toluene is by a dioxygenase enzyme system to form (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene(cis-toluene dihydrodiol) which is oxidized to 3-methylcatechol which is further degraded by enzymes of a meta cleavage pathway.
A third catabolic pathway for the degradation of toluene has been recently identified in PmKR1. It has been found that PmKR1 catabolizes toluene by a novel pathway which is completely different than either of the two pathways described above. Richardson and Gibson, Abst. Ann. Meet. Am. Soc. Microbiol. K54:156 (1984). The catabolic pathway for the degradation of toluene by PmKR1 has been designated TMO, because the first step in the pathway is catalyzed by a unique enzyme complex, toluene monooxygenase. The biochemistry of the enzymes and proteins of this pathway has been recently studied in detail by Whited, Ph. D. Dissertation, The University of Texas at Austin, Library Reference No. W586 (1986).
A brief summary of the TMO pathway in PMKR1 is as follows: in the initial step toluene is oxidized to p-cresol, followed by methyl group oxidation to form p-hydroxybenzoate, followed by hydroxylation to protocatechuate and subsequent ortho ring cleavage. The steps of the TMO pathway as outlined by Whited (supra) are diagrammed in FIG. 1. In the first step of the TMO pathway, toluene is converted by toluene monooxygenase to p-cresol. PmKR1 elaborates a unique multicomponent enzyme system which catalyzes this first step monooxygenase reaction. According to Whited, (supra), at least three protein components are involved: oxygenase (at least 2 subunits of 50,000 d. and 32,000 d.), ferredoxin (23,000 d.) and NADH oxidoreductase (molecular weight unknown).
At present, despite the substantial advances in the understanding of the biochemistry of the enzymes and proteins of the TMO pathway and beginning genetic studies (Yen et al. Abstract, University of Geneva EMBO Workshop, Aug. 31-Sept. 4, 1986), the art has not been provided with information regarding the genes encoding the enzymes and proteins of the toluene monooxygenase system in PmKR1 or the usefulness of such genes and gene products in certain microbial bioconversions. The art has also not been provided with microorganism host cells containing novel recombinant plasmids containing PmKR1 toluene monooxygenase genes, in which certain of these microorganism host cells express toluene monooxygenase enzyme activity at levels that exceed the activity of wildtype PmKR1 cells.